Hypoxic pulmonary vasoconstriction (HPV) serves as an important regulatory mechanism maintaining adequate arterial oxygenation in response to hypoxia, but can also result in pulmonary hypertension. While the mechanism underlying HPV is incompletely understood, an increase in intracellular calcium concentration ([Ca2+]i) in smooth muscle cells plays a critical role in the development of HPV. The hypoxic [Ca2+]i increase is due to Ca2+ release from the sarcoplasmic reticulum (SR), as well as Ca2+ influx through voltage-dependent Ca2+ channels. The hypoxic Ca2+ influx is likely caused by inhibition of K+ currents and activation of Ca2+-activated Cl- currents. Both hypoxic effects may be secondary to Ca2+ release. However, the molecular processes coupling hypoxia to Ca2+ release remain elusive. We and other investigators have shown that Ca2+ release following hypoxia or metabolic inhibition is blocked by depletion of the SR Ca2+. Our preliminary data indicate that inhibition of the NADPH oxidase blocks hypoxic [Ca2+]i increase, and that H2O2 reverses hypoxic potentiation of agonist-induced [Ca2+]i rise and slowing of calcium decay. We have also found that reducing agents mimic the hypoxic Ca2+ release. Therefore, in this application we will address the following questions (specific aim): (1) does hypoxic Ca2+ release occur through ryanodine receptors, inositol triphosphate receptors or both? (2) do recently discovered endogenous Ca2+ releasing mediators such as cyclic ADP-ribose, nicotinic acid adenine dinucleotide phosphate and FK506 binding protein mediate hypoxic Ca2+ release? (3) is the NADPH oxidase a primary oxygen sensor in hypoxic Ca2+ release? (4) do H2O2, intracellular reducing agents and protein kinase C serve as signal transducers in the hypoxic Ca2+ release? These aims will be pursued by using simultaneous measurements of membrane currents and whole-cell or local [Ca2+]i (Ca2+ sparks) in single voltage-clamped pulmonary resistance artery myocytes. Transgenic mice, gene overexpression and inhibition of gene expression will be also used to define the coupling of hypoxia to Ca2+ release. The findings of this proposed research will extend our understanding of cellular and molecular mechanisms responsible for the development of HPV, and may lead to identify a potential novel target to treat HPV.